cold-regulated cereal chloroplast lea-like proteins. molecular … · 2002. 6. 20. ·...

Cold-Regulated Cereal Chloroplast LEA-Like Proteins.Molecular Characterization and Functional Analyses

Christian NDong, Jean Danyluk, Kenneth E. Wilson, Tessa Pocock, Norman P.A. Huner, andFathey Sarhan*

Departement des Sciences biologiques, Universite du Quebec a Montreal, C.P. 8888 Succursale Centre-ville,Montreal, Quebec, Canada H3C 3P8 (C.N., J.D., F.S.); Department of Molecular Biology, University ofGeneva, 30 Quai Ernest-Ansermet, CH–1211 Geneva 4, Switzerland (K.E.W.); and Department of PlantSciences, University of Western Ontario, London, Ontario, Canada N6A 5B7 (T.P., N.P.A.H.)

Cold acclimation and freezing tolerance are the result of complex interaction between low temperature, light, andphotosystem II (PSII) excitation pressure. Previous results have shown that expression of the Wcs19 gene is correlated withPSII excitation pressure measured in vivo as the relative reduction state of PSII. Using cDNA library screening and datamining, we have identified three different groups of proteins, late embryogenesis abundant (LEA) 3-L1, LEA3-L2, andLEA3-L3, sharing identities with WCS19. These groups represent a new class of proteins in cereals related to group 3 LEAproteins. They share important characteristics such as a sorting signal that is predicted to target them to either thechloroplast or mitochondria and a C-terminal sequence that may be involved in oligomerization. The results of subcellularfractionation, immunolocalization by electron microscopy and the analyses of target sequences within the Wcs19 gene areconsistent with the localization of WCS19 within the chloroplast stroma of wheat (Triticum aestivum) and rye (Secale cereale).Western analysis showed that the accumulation of chloroplastic LEA3-L2 proteins is correlated with the capacity of differentwheat and rye cultivars to develop freezing tolerance. Arabidopsis was transformed with the Wcs19 gene and the transgenicplants showed a significant increase in their freezing tolerance. This increase was only evident in cold-acclimated plants. Theputative function of this protein in the enhancement of freezing tolerance is discussed.

Cold acclimation is a process that occurs in manytypes of organisms in response to a decrease in tem-perature (Levitt, 1980; Graumann and Marahiel, 1996;Hughes and Dunn, 1996; Huner et al., 1998). Thephysiological, biochemical, and molecular processesinvolved in the attainment of cold acclimation havebeen studied extensively, but a complete understand-ing of the functions of the various genes induced bylow temperature is still lacking (Thomashow, 1999).However, the role of some of these proteins has beendetermined, and they act as transcription or elonga-tion factors, antifreeze proteins, and proteins in-volved in stabilizing membrane architecture (Jonesand Inouye, 1994; Nishida and Murata, 1996; Chun etal., 1997; Gilmour et al., 1998).

At low temperatures, organisms have two primarydifficulties. The first problem is maintaining mem-branes in a fluid state that allow them to resistsubzero temperatures (Nishida and Murata, 1996).Membrane integrity and cell survival can also becompromised because of both intracellular and extra-

cellular ice formation (Steponkus, 1984; Thomashow,1999; Yu and Griffith, 1999). The second problem isencountered by photosynthetic organisms and is re-lated to the thermodependency of photosyntheticelectron transport and carbon fixation, which areslowed at low temperature (Guy, 1990; Huner et al.,1998). However, the primary photochemical reac-tions of light absorption by the light-harvesting an-tennae and the transfer of excitation energy to thephotosynthetic reaction centers occur at rates that areessentially independent of temperature. Primarycharge separation and exciton transfer from the an-tennae pigments to the reaction centers can still occurat the temperature of liquid N2 (2196°C; Butler,1978). Thus, changes in temperature can result in theinability of the organism to use the absorbed lightenergy, leading to the over-reduction of the electrontransport chain (Huner et al., 1998). This can, in turn,lead to photoinhibition of photosystem II (PSII) andincreased production of oxygen radicals (Asada,1994). In chloroplasts, there is a dynamic equilibriumbetween PSII damage and repair. When the rate ofdamage is greater than the rate of repair, photoinhi-bition occurs and is reflected in a decrease in themeasurable FV/FM. Thus, avoidance of photoinhibi-tion can occur either by decreased rates of damageor increased rates of repair (Melis, 1999). Cold-acclimated cereals have been shown to be less sensi-tive to photoinhibition, and interestingly, this ap-pears to correlate with the maximum freezing

1 This work was supported by research grants from NaturalSciences and Engineering Research Council of Canada and Fondspour la Formation de Chercheurs et l’Aide a la Recherche (to F.S.and N.P.A.H.).

* Corresponding author; e-mail [email protected]; fax514 –987– 4647.

Article, publication date, and citation information can be foundat www.plantphysiol.org/cgi/doi/10.1104/pp.001925.

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tolerance of the plants (Hurry and Huner, 1992; Grayet al., 1997). This resistance has in part been ex-plained by decreased rates of damage due to in-creased photosynthetic capacity (Hurry and Huner,1992).

Previous screening of a wheat (Triticum aestivum)cold-acclimated cDNA library allowed the identifica-tion of a novel cold-regulated gene called Wcs19 thatencodes a protein of unknown function (Chauvin etal., 1993). The mRNA of this gene was shown to beexpressed exclusively in photosynthetic tissues, andits transcript accumulation was found to be corre-lated with the level of PSII excitation pressure (Grayet al., 1997). Therefore, this gene was not regulated bytemperature per se but rather by a complex interac-tion of temperature and light. Sequence comparisonshave shown the existence of closely related genes inbarley (Hordeum vulgare; cor14b; Crosatti et al., 1999)and wheat (Wcor14a and Wcor14b; Tsvetanov et al.,2000), suggesting that Wcs19 may be part of a smallfamily of genes with similar or overlapping functionsand regulation. In an effort to gain a better insightinto the structural and functional features of thisgene family, we first set out to identify and charac-terize Wcs19 homologs and orthologs in rye (Secalecereale) and wheat. Second, given the correlation be-tween Wcs19 mRNA accumulation and PSII excita-tion pressure, we developed antibodies to the WCS19protein, and used them to examine its accumulationand subcellular localization in rye and wheat in re-sponse to excitation pressure and cold acclimation.These molecular and biochemical analyses revealedthat this new gene family encodes chloroplastic pro-teins related to group 3 LEA (late embryogenesisabundant) proteins. Last, in an attempt to begin de-termining the function of this protein family, Arabi-dopsis was transformed with the Wcs19 gene, a rep-resentative member of this family, under the controlof a constitutive promoter. The effects of the consti-tutive expression of WCS19 on the freezing toleranceand resistance to photoinhibition of transgenic Ara-bidopsis leaves are described and discussed withrespect to the predicted structural characteristics ofthe WCS19 protein and its orthologs.

RESULTS

Identification of Wcs19 Homologous Genes

Sequence analyses have indicated that WCS19shares two regions of homology with BCOR14b pro-tein from barley (Crosatti et al., 1999) and WCOR14aprotein from wheat (Tsvetanov et al., 2000). The firstconserved region (I) lies at the N-terminal end ofthese proteins, whereas the second conserved region(II) lies at the C-terminal end of these proteins (Fig.1). Between these conserved regions exists a variableregion (V) that shows little identity (Fig. 1). Theseanalyses suggested that WCS19 might be part of asmall family of related proteins. The first strategy

allowed us to identify three new genes, Rep14, Rep13,and Wcor14c (Fig. 1) that are expressed during growthat high levels of PSII excitation pressure and duringcold acclimation. Based on their sequences and theirdegree of homology, these small proteins and thepreviously isolated WCOR14a, BCOR14b, andWCS19 can be classified into two groups. The firstgroup contains WCOR14a, WCOR14c, REP13, andBCOR14b (Fig. 1), and members of this group showidentities ranging from 63% to 95%. The secondgroup contains WCS19, REP14; and BF625247 (Fig. 1);these proteins show identities ranging from 89% to95%. However, when proteins from the first groupare compared with proteins from the second group,the percent identity falls to between 46% and 55%.

Results from our second strategy revealed thatboth groups of proteins share significant homologieswith a new barley protein BG369977n reconstitutedfrom several overlapping expressed sequence tags(ESTs; Fig. 1). This large 293-amino acid protein wasfound to be more homologous to BCOR14b (58%identity and 79% similarity) than to BF625247 (45%identity and 68% similarity). The main difference isthe variable region that extends for 191 amino acidsin BG369977n compared with 50 in BCOR14b (Fig. 1).Moreover, sequence comparisons suggest that thislarge variable region shares a homology with severalproteins that belong to group 3 LEA proteins. Carefulanalyses of the BG369977n protein shows that itcould have as many as 14 imperfect copies of the11-mer repeat that characterize group 3 LEA pro-teins (Dure, 1993; Fig. 1). Furthermore, the identifi-cation of two partial wheat ESTs homologous toBG369977n may suggest the existence of a thirdgroup of related proteins in the three grass species.Based on sequence analyses of the three groups andtheir relation to group 3 LEA proteins, they arenamed LEA3-L1 (WCOR14a, WCOR14c, REP13, andBCOR14b), LEA3-L2 (WCS19, REP14, and BF625247),and LEA3-L3 (BG369977n). LEA3-L stands for LEAgroup 3 protein-like.

Arabidopsis genome was also found to encode twolarge proteins, T10644 (266 amino acids) andBAB10116 (331 amino acids) that have several 11-merrepeats and, thus, may represent Arabidopsis ho-mologs of the LEA3-L3 BG369977n protein. Thesetwo proteins share 31% to 36% identity and 59%similarity with the LEA3-L3 protein BG369977n frombarley. Higher identities between the C-terminal re-gions of these two Arabidopsis proteins and theLEA3-L3 BG369977n were found (44%–50%) suggest-ing that this region may have important conservedproperties for the function of these related proteins.Therefore, only the alignment of the conservedC-terminal regions from members of the threeLEA3-L groups present in grass species and the twoArabidopsis proteins is presented in Figure 2A. Inaddition, four other proteins were found to have this

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highly conserved C-terminal region and were in-cluded in the alignment (Fig. 2A).

Identification of Chloroplast and MitochondriaSorting Signals

To determine the properties of the N-terminal end(region I) of the LEA3-L1, -L2, and -L3 polypeptides

and their relationship with other proteins presentedin Figure 2A, all polypeptides were analyzed withTargetP, Predotar, ChloroP, and Mitoprot softwares.The output from TargetP incorporates a measure ofaccuracy in the prediction, which was shown to varyin test cases from 99% accuracy with reliability class(RC) 5 1% to 55% with RC 5 5 (Emanuelson et al.,2000). Using this program, the LEA3-L1 (RC 5 3) and

Figure 1. Alignment of three groups of proteins from wheat, rye, and barley sharing identities with WCS19. ClustalWalignment of wheat WCS19 (accession no. L13437), rye REP14 (accession no. AF491840), barley BF625247 (deduced fromcv Morex; EST accession no. BF625247), wheat WCOR14a (accession nos. AF207545 and AF491838) and WCOR14c(accession no. AF491837), rye REP13 (accession no. AF491839), and barley BCOR14b (accession no. M60732) andBG369977n. ESTs from barley cv Morex, BG369977, BG369422, BE454426, and BE196464 were used to generate thesequence BG369977n of 999 bases. The sequence contained an open reading frame with three possible start codons andone stop codon encoding a protein of 293 amino acids. Residues within motifs resembling the consensus 11-mer repeatcharacteristic of group 3 LEA proteins are shown and numbered above the BG369977n protein. { distinguishes the groupsof proteins. I, The conserved region coding for the putative signal peptide; II, the conserved C-terminal region; V, the variableregion. The arrow indicates the putative cleavage site of the signal peptide determined with ChloroP. The boxed amino acidsin region I highlight a sequence resembling the 14-3-3 recognition motif. Shaded amino acids in the largely a-helical matureproteins represent regions predicted to adopt a coil conformation by PELE, a program that uses eight different algorithms tostudy secondary structure. 2, Gaps introduced to maximize alignment; *, identical residues; :, highly conserved residues;., conserved residues.

Cold-Regulated Chloroplast Late Embryogenesis Abundant Proteins

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Figure 2. Alignment and structure analysis of the conserved C-terminal region from different plants. A, ClustalW alignmentand Multicoil analysis of the conserved C-terminal region (II) of proteins from the LEA3-L1, -L2, and -L3 groups with sixproteins from other plants sharing a homology in this region. Arabidopsis proteins of 331 amino acids (BAB10116) and 266amino acids (T10644) were deduced from genomic sequencing. PM32 (AF166485) is a 173-amino acid maturation proteinidentified in soybean (Glycine max; Chow et al., 1999). L42465 is a 190-amino acid LEA protein identified in Picea glauca(Dong and Dunstan, 1996). AU089534n is a 165-amino acid protein from Lotus japonicus that was reconstituted from threeESTs: AU089534, AU089020, and AU089581. BE592220n is an incomplete protein (102 amino acids) from sorghum(Sorghum bicolor) reconstituted from ESTs BE592220 and BE592752. The consensus sequence represents the compilationof amino acids conserved in at least 11 out 14 proteins. To the right of the alignment is a summary of the probabilities forthe C-terminal region of the 14 proteins to form a coiled coil motif. The results were obtained using Multicoil scores basedupon pair wise interactions for residues at distances 2, 3, and 4 apart with the dimeric table and at distances 3, 4 and 5 apartwith the trimeric table (Wolf et al., 1997). Segment and minimum probability (%), High scoring segments with minimum 25residues are defined by numbering relative to the first residue in the C-terminal region alignment. In this segment, the lowesttotal probability for a residue or subsegment to form a coiled coil motif is given as a percentage. Maximum total probability,The maximum probability for a residue or subsegment in the previously defined 25 residues segment. Trimeric oligomer-ization ratio, The trimeric score divided by the total score in the C-terminal region. To analyze L42465 from P. glauca, thePro (a helix breaker) at position 10 was changed to Ala the most common amino acid at that position. B, Helical wheelprojection of the consensus sequence generated from region II of the 14 proteins. Amino acids and the number of times theywere repeated in at least 11 proteins are indicated for each position. Circled positions indicate single residues conserved inat least 11 proteins. *, Position 1 in Figure 2A.

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-L2 (RC 5 2) groups were predicted to be targeted tothe chloroplast, whereas the LEA3-L3 protein (RC 54) was predicted to be targeted to the mitochondria.Predictions using Predotar follow the same patternwith localization probabilities for the LEA3-L1 group(chloroplast P 5 0, 469), L2 group (chloroplast P 5 0,924) and L3 protein (mitochondrion P 5 0, 708).Because chloroplast and mitochondria sorting signalsshare common properties (Emanuelson et al., 2000),programs such as ChloroP and Mitoprot predicted,respectively, a chloroplast and mitochondria local-ization for these three groups of proteins. Analysis ofthe Arabidopsis, P. glauca, and L. japonicus proteins(Fig. 2A) also revealed that they are likely targeted tothe chloroplast and/or the mitochondria. This anal-ysis suggests that the proteins presented in Figure 2Ashare an additional important feature, which is asorting signal predicted to target them to organelles.

Secondary and Tertiary Structure Predictions for theMature Proteins

The mature proteins of the LEA3-L1 and L2 groups(Fig. 1) share several characteristics such as similarsize (8.5–9.6 kD) and acidic pI (4.1–4.5). The analysesof these proteins by PELE reveals that the variableregion V is predicted to contain segments in the coilconformation (Fig. 1; shaded amino acids). Thesesegments may represent breaks in the largelya-helical proteins and, thus, induce slightly differentproperties in both groups. However, biochemical ev-idence is needed to support these predictions.

To get a better idea of the structural features orproperties of these proteins, a consensus helicalwheel projection was generated for the conserveda-helical region II of all proteins presented in Figure2A. Analysis of the projection in Figure 2B reveals a140o hydrophobic side that is bordered on either side(40 and 60o) by basic or polar amino acids. In the 40o

border region, only one protein in 14 contained anacidic amino acid, whereas in the 60o region, twoproteins contained an acidic amino acid. It is impor-tant to note that the majority of positions showing ahigh identity in the consensus C-terminal region areconcentrated to one side of the amphipatic a-helix.

The strict conservation of amphipatic character andidentity in certain amino acid positions in theC-terminal end raised the possibility that this regionmay be involved in some sort of tertiary interaction.One type of structure that is known to require anamphipatic property is the coiled coil motif (Lupas etal., 1991; Berger et al., 1995; Wolf et al., 1997). Thus,proteins containing the conserved C-terminal endwere analyzed using the program Multicoil (Wolf etal., 1997). This program helps in identifying thecoiled coil motif in proteins and the oligomerizationstates. The results of this analysis are presented inFigure 2A and show that seven out 14 proteins havesegments with minimum total probability scores

ranging from 28% to 50%, with most of the probabil-ity coming from the trimeric score indicating a trim-eric oligomerization. Overall, this analysis revealsthat homologs from different species share distinctproperties such as an amphipatic character and aconserved C-terminal region that may be involved inoligomerization.

Expression and Chromosomal Localization ofLEA3-L1 and LEA3-L2 Genes

The results in Figure 3A illustrate the RNA hybrid-ization blot for the 600-bp transcript correspondingto the rye LEA3-L2 (Rep14) gene. The blot for the ryeLEA3-L1 (Rep13) gene gave identical results (resultnot shown). Both genes appear to be regulated byPSII excitation pressure because they exhibitedhigher expression in rye plants grown at 20/800 for14 d or 5/250 for 40 d compared with plants grown at20/250 or 5/50 (Fig. 3A). The expression and regu-lation of rye LEA3-L1 and -L2 by PSII excitationpressure are consistent with previous reports for thewheat Wcs19 gene (Chauvin et al., 1993; Gray et al.,1997).

The ditelocentric series of Chinese Spring wheat inwhich one homologous pair of chromosome arms ismissing in each line, was used to determine whichchromosome arms carry members of the LEA3-L1and LEA3-L2 groups (results not shown). Resultsusing this series have shown that: (a) wheat LEA3-L1and L2 genes reveal a different pattern of genomicorganization indicating that these two genes are suf-ficiently different to be used as probes for localiza-tion; (b) the wheat LEA3-L1 genes were mapped tothe long arms of chromosome 2, as was Bcor14b(Vagujfalvi et al., 2000); and (c) the wheat LEA3-L2genes could not be mapped with the available geneticstocks.

Accumulation of LEA3-L2 Protein (WCS19) duringExposure to High Light and Cold Temperature

To determine whether the LEA-L2 (REP14) proteincould remain soluble upon boiling, protein extractsfrom 5/250 rye grown leaves were boiled for 30 min.Immunoblot results in Figure 3B indicate that theREP14 protein remained soluble as found in all LEAproteins. Although the anti-WCS19 antibody wasraised against a LEA3-L2 protein, we cannot rule outthe possibility that this immune serum also cross-reacts partially with proteins from the LEA3-L1group. However, the anti-WCS19 antibody does notseem to cross-react with LEA3-L3 proteins becauseno additional bands were detected.

The immune serum was also used to measure theaccumulation kinetics of proteins during exposure tohigh-excitation pressure conditions induced by highlight (20/800) and low temperature (5/250). Whenrye plants grown at 20/250 were shifted to high-





excitation pressure conditions, the anti-WCS19 anti-body detected a 14-kD protein that accumulatedgradually and reached a maximum level near 12 d at20/800 or 10 d at 5/250 (Fig. 3, C and D). The 14-kDprotein, which may represent the mature protein, isnot in agreement with the calculated molecular mass(9.6 kD) deduced from the mature rye REP14 protein.Such differences have been observed previously withdifferent plant stress proteins (Houde et al., 1992a).

In addition, the accumulation level of LEA3-L2proteins during low temperature exposure in a num-ber of cultivars differing in their capacity to coldacclimate was evaluated. The results in Figure 3Eshow that LEA3-L2 proteins accumulate to a higherlevel in cultivars with an enhanced capacity to de-velop freezing tolerance. The more freezing tolerantwheat cv Bes, CNN, and Ulian (LT50 of 216.4, 219,and 219.5°C, respectively) maintained a higher levelof WCS19 protein compared with the less freezingtolerant cv Glen (25.5°C), Man (26.2°C), and CS(29.4°C). A similar result was obtained with thefreezing tolerant rye cv Puma (224.8°C) comparedwith rye cv Gaz (26.5°C). These results support theconcept that LEA3-L2 proteins are associated withthe plant capacity to develop freezing tolerance.

Localization and Immunolocalization ofLEA3-L2 Proteins

The results in Figure 4A show that the wheatLEA3-L2 protein (WCS19) accumulates specifically inthe leaves of wheat during cold acclimation. No sig-nal was detected in the roots nor in the crown, sug-gesting that WCS19 accumulates exclusively in thephotosynthetic tissue. In addition, analysis of total

Figure 3. Expression analysis and accumulation of LEA3-L2 proteinsin rye and wheat. A, Transcript accumulation of rye LEA3-L2 geneusing Rep14 as probe. Equal amounts of total RNA (5 mg) wereseparated by agarose gel electrophoresis in the presence of formal-dehyde and transferred to a nitrocellulose membrane. 20/800, 20/250, and 20/50 represent rye plants grown at 20°C and at

800 mmol m22 s21, 250 mmol m22 s21, or 50 mmol m22 s21,respectively. 5/250 and 5/50 represent rye plants grown at 5°C and at250 mmol m22 s21 or 50 mmol m22 s21, respectively. The size ofRep14 transcript is indicated on the right in bases. B, Boiling solu-bility of LEA3-L2 proteins. NA and A, Soluble proteins (5 mg) fromleaves of rye plants (cv Musketeer) grown at 20/250 (24 d) and 5/250(40 d), respectively. In addition, soluble proteins from 5/250 leaveswere heated at 100°C for the time indicated. Insoluble (I) and boilingsoluble (S) fractions were analyzed by immunoblotting as describedin “Materials and Methods.” The size of the mature REP14 in kilo-daltons is indicated at right. C, Accumulation kinetics of the ryeLEA3-L2 protein during high-light exposure. Rye plants grown at20/250 for 24 d were shifted to 20/800 conditions for the number ofhours (h) and days (d) indicated. Soluble proteins (5 mg) were ana-lyzed by immunoblotting. To show uniform loading, the CoomassieBlue-stained Rubisco subunit (55 kD) is shown. D, Accumulationkinetics of the rye LEA3-L2 protein during low-temperature acclima-tion. Plants grown at 20/250 for 24 d were shifted to 5/250 conditionsfor the time indicated, and soluble proteins (5 mg) were analyzed byimmunoblotting. E, Accumulation of LEA3-L2 proteins in differentwheat and rye cultivars cold acclimated for 49 d. Immunoblot anal-ysis was done with soluble proteins (5 mg) from leaf tissues of springrye and wheat cv Gazelle (Gaz), Glenlea (Glen), Manitou (Man), andChinese Spring (CS) and from winter rye and wheat cv Puma (Puma),Besostoya (Bes), Cheyenne (CNN), and Ulian (Ulian).

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chloroplast extracts and stromal and thylakoid frac-tions from cold-acclimated wheat shows that theWCS19 protein accumulates specifically in thestroma (Fig. 4B).

To further confirm whether LEA3-L2 proteins weretruly localized to the chloroplast, we used immuno-cytochemical localization. When sections of leaf tis-sues from control rye plants grown at 20/250 wereincubated with the anti-WCS19 antibody and withgold-conjugated antiserum to rabbit immunoglobu-lins, little deposition of gold particles was observedin the chloroplast (0.49 6 0.09 gold particles mm22 forn 5 10 chloroplasts counted; Figs. 5C and 6). On theother hand, leaf sections from rye plants grown at5/250 revealed the strongest deposition of gold par-ticles in the chloroplast (1.38 6 0.13 gold particlesmm22 for n 5 10 chloroplasts counted; Figs. 5A and6). In addition to being present in the chloroplaststroma, the rye LEA3-L2 protein (REP14) was alsofound to be associated with the periphery of thethylakoid membranes but not membrane bound (re-sult not shown). Finally, leaf sections from plantsgrown at 20/800 revealed an intermediate level ofdeposition of gold particles in the chloroplast (0.82 60.09 gold particles mm22 for n 5 10 chloroplastscounted; Figs. 5B and 6).

To confirm the specificity of chloroplast labelingobtained with the anti-WCS19 antibody, we con-ducted similar experiments using sections of 20/800grown leaves, which were immunolabeled with an-tibodies raised against the LHCII polypeptides ofspinach (Spinacia oleracea) or the Rubisco holocom-plex of rye (results not shown). Results confirmed theintegrity of the fixed tissue because it was possible toobserve that the Rubisco immunogold label was pri-marily associated with the stromal compartment,whereas the LHCII immunogold label was primarilyassociated with the thylakoid membranes of the chlo-roplast. Additional control tests including the use ofpreimmune serum or distilled water did not revealany deposition of gold particles.

Thus, it appears that LEA3-L2 is either localized tothe chloroplast stroma or loosely associated with thy-lakoid membranes, which confirms the results of thebiochemical fractionation. Therefore, members fromboth groups, rye and wheat LEA3-L2 and barley

Figure 5. Electron microscopy of rye leaf sections incubated withanti-WCS19 antibody. A, Plants grown at low temperature for 40 d(5/250). B, Plants grown at high light for 14 d (20/800). C, Nonac-climated plants grown at 20/250 for 24 d. The magnification of eachimage is 22,0003. The images shown are representative of typicalWCS19 labeled chloroplasts from plants grown under the givenconditions.

Figure 4. Wheat LEA3-L2 (WCS19) tissue distribution and subcellu-lar localization. A, Immunoblot analysis of soluble leaf proteins (5mg) present in different tissues of wheat (cv Fredrick) grown underlow-temperature conditions. NA, Nonacclimated plants; A, plantscold acclimated for 2 weeks. B, Immunoblot analysis of proteins (5mg) present in different chloroplast compartments. Legend as in A.





LEA3-L1 protein (BCOR14b; Crosatti et al., 1999), arenow known to specifically accumulate in the stroma.This suggests that both groups of proteins have afunctional role in the chloroplast during acclimationto growth at high-excitation pressures induced byhigh light and/or low temperature.

Effects of the Constitutive Expression of the WheatLEA3-L2 (WCS19) on Cold Acclimation and FreezingTolerance in Arabidopsis

Although it is well documented that the accumu-lation of LEA3-L2 mRNAs and proteins is increasedin response to cold temperature, it is still not knownwhat function these proteins might have in the coldacclimation process. Using Arabidopsis plants thathave one representative of the LEA3-L2 genes(Wcs19) inserted under the control of the cauliflowermosaic virus (CaMV) 35S promoter, we examined thefreezing tolerance of plants grown at 20°C and plantsthat had been grown at 20°C but shifted to lowtemperature (5°C) for 7 d. To ensure that the proteinwas being expressed, soluble leaf protein extracts ofthe three transgenic lines (B, C48, and C71) were an-alyzed by immunoblots. It can be observed from thedata in Figure 7 that, whereas the level of the WCS19protein present in Arabidopsis transgenic plants islower than in cold-acclimated (5/250) grown rye orwheat, the protein is present in transgenic plants butabsent in wild-type plants. The freezing tolerance ofArabidopsis leaves was determined using the elec-trolyte leakage technique and reveals that in nonac-climated plants, the LT50 values for the transgenicplants were similar to wild-type plants, indicating noincrease in the freezing tolerance. The LT50 of wild-type leaves was 24.0 6 0.4°C compared with 23.5 60.3°C in transgenic leaves (line B), 24.0 6 0.5°C for

line C48, and 24.6 6 1.4°C for line C71 (Fig. 8A).However, when the freezing tolerance of the cold-acclimated leaves (plants shifted from 20°C to 5°C for1 week) was compared, the transgenic lines weresignificantly more freezing tolerant. The LT50 ofleaves from wild-type plants shifted to 5°C for 1week was 25.9 6 0.4°C compared with 27.2 6 0.5°Cfor line B, 27.9 6 0.3°C for line C48 and 28.1 6 0.3°Cfor line C71 (Fig. 8B). These differences were statisti-cally significant (P 5 0.02; 0.0018 and 0.0013 two-tailed Student’s t test respectively for line B, C48 andC71). Moreover, when the LT50 values of the threetransgenic lines are combined, the transgenic groupremained statistically more freezing tolerant (27.7 60.39 P 5 0.008) compared with the wild type. Thus, itappears from the results obtained that the WCS19protein may play a role in the cold acclimation pro-cess and that its constitutive expression could en-hance freezing tolerance.

Effects of the Constitutive Expression of the WheatLEA3-L2 (WCS19) on the Tolerance of ArabidopsisPlants to Photoinhibition

Cold acclimation is correlated quite strongly withincreased tolerance to photoinhibition, and maxi-mum LT50 is dependent upon low temperature andhigh-light levels (Oquist et al., 1993; Gray et al., 1997;Pocock et al., 2001). Therefore, because wheat and ryeLEA3-L2 proteins are localized in the chloroplast,and their transcript abundance is regulated in re-sponse to the redox-state of PSII, we examinedwhether the presence of the WCS19 protein in theleaves of transgenic Arabidopsis plants resulted inincreased resistance to photoinhibition.

Detached leaves of Arabidopsis plants that hadbeen grown at 20°C or grown at 20°C and transferredto 5°C were exposed to 1,600 mmol photons m22 s21

for 3 h. FV/FM was determined at 30-min intervals toestimate the level of photoinhibition of PSII. Thepresence of the WCS19 protein in the leaves of plants

Figure 6. Density of the immunogold labeling obtained with theanti-WCS19 antibody in chloroplasts of rye plants exposed to threegrowth conditions. The number of gold particles per square micro-meter of the chloroplasts was determined using the Northern EclipseImage Analysis software package. This allowed the analysis of digi-tized images of representative chloroplasts from WCS19 labeledsections of rye leaves grown under the indicated conditions. Barsrepresent mean 6 SE, n 5 10.

Figure 7. Accumulation of the wheat LEA3-L2 (WCS19) protein intransgenic Arabidopsis grown at 20°C/100 mmol m22 s21. Solubleproteins (5 mg) from rye leaves (5/250) were used as positive control.The immunoblot was overexposed to show that the anti-WCS19antibody does not recognize any endogenous proteins in wild-type(WT) Arabidopsis plants. B, C48, and C71 represent the transgeniclines. NA, Nonacclimated; A, plants cold acclimated for 1 week.

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grown at 20°C had no apparent effect on the toler-ance to photoinhibition when wild-type and trans-genic lines are compared (Fig. 9A). The leaves of bothwild-type and transgenic plants were photoinhibitedto the same degree, the dark adapted FV/FM after3 h at photoinhibitory conditions was reduced to35.5% 6 1.9% of the initial value for wild-type plantscompared with 31.4% 6 2.3% for line B, 34.8% 6 1.7%for line C48, and 37% 6 1.6% for line C71. However,when the plants had been shifted from 20°C to 5°Cfor 1 week, the presence of the WCS19 protein didappear to indicate minimal but significant protection(P 5 0.03, two-tailed Student’s t test) of the leaves ofthe C71 line only from photoinhibition after 3 h (Fig.9B). In leaves of wild-type plants, FV/FM was de-creased to 31% 6 2.1% compared with 36.3% 6 5.4%for line B, 37.3% 6 3.8% for line C48 and 43.5% 6 3.9%for line C71.

DISCUSSION

Results presented in this report show that grassspecies such as rye, wheat, and barley contain at leastthree different groups of LEA3-like proteins that canbe classified as small (LEA3-L1 and LEA3-L2 groups)and large (LEA3-L3 group) proteins. These threegroups and their related proteins in other plantsshare several common characteristics. An importantfeature of these proteins is the presence of a sortingsignal that is predicted to target them either to thechloroplast, mitochondria or both. Although a fewcases are known where one sorting signal can routewith similar efficiency a protein to chloroplast andmitochondria (Creissen et al., 1995; Chow et al., 1997;Akashi et al., 1998; Menand et al., 1998), such dualtargeting signals are predicted to be quite rare(Emanuelson et al., 1999). Detailed analysis of thesorting signals present in the LEA3-L1, -L2, and -L3groups of proteins (Fig. 1) revealed that the LEA3-L2proteins contain a sequence sharing a high identity

Figure 8. Effect of the constitutive expression of wheat LEA3-L2(WCS19) on the freezing tolerance of Arabidopsis leaves. A, Electro-lyte leakage curves from leaves of plants grown at 20°C. B, Electro-lyte leakage curves from leaves of plants shifted from 20°C to 5°C for1 week. Wild-type (E) and transgenic lines B, C48, and C71 (Œ, f, andl, respectively). From the electrolyte leakage curves, the LT50 wasdetermined as the temperature at which 50% of the ions leaked outthe leaf and used as an estimate of freezing tolerance. The experi-ments were done four times (n 5 3 leaves for each temperature) foreach transgenic lines and values represent mean 6 SE.

Figure 9. The effect of constitutive expression of the wheat LEA3-L2(WCS19) protein on photoinhibition of Arabidopsis leaves. A, Pho-toinhibition of detached leaves from plants grown at (20°C). B,Photoinhibition of detached leaves from plants shifted from 20°C to5°C for 1 week. Symbols as in Figure 8. The experiments were donefour times (n 5 3 leaves for each time) for each transgenic lines andvalues represent mean 6 SE. Where not visible, the error bars aresmaller than the symbols.





with the 14-3-3 recognition motif (R S X S/T X P; Mayand Soll, 2000; Fig. 1). On the other hand, this se-quence is less conserved in the LEA3-L1 group andnonexistent in the LEA3-L3 group. It was shown thatwhen this recognition motif was phosphorylated, itinteracts with 14-3-3 proteins (May and Soll, 2000).This complex-bound precursor was much more effi-cient for import into isolated chloroplasts than wasthe free precursor protein (May and Soll, 2000). Thehigh identity between these three groups sorting sig-nals suggest that they may have diverged from acommon ancestor rather recently, and/or some pri-mary sequence is being conserved because of somefunction other than just directing import to the chlo-roplast. The fact that, to date, most of the newlyidentified members of this new class of LEA3-likeproteins are predicted to be targeted to organelles isintriguing and suggests that properties and functionsinferred from their sequences may have evolved towork in a specific organelle environment. Understress conditions, it will be critical to protect thechloroplasts and mitochondria to maintain cellularenergy production. Thus, these groups of proteinscould be very important for stress survival of the celland/or organism.

These proteins differ on at least one aspect, whichis the length of the sequence between their sortingsignal and the conserved C-terminal region. Thisvariable region (V) shares a similarity with group 3LEA proteins, and allowed us to classify these pro-teins as distantly related to this group. A main char-acteristic of group 3 LEA proteins is the presence ofseveral copies of an 11-mer repeat (Dure, 1993). Pro-teins containing several copies of this repeat havebeen proposed to function by scavenging ions duringdesiccation (Dure, 1993). It should be noted that atleast another function for group 3 LEA proteins hasbeen inferred, because it was shown that a proteincontaining a sufficient number of the 11-mer repeathad some cryoprotective activity (Honjoh et al.,2000). Because the smaller LEA-like proteins identi-fied in this study contain little if any of the 11-merrepeats, it is doubtful that they are involved in scav-enging ions. This raises an interesting dilemma con-cerning their evolution, suggesting that they evolvednew functions or that there is a common, yet un-known, function of group 3 LEA proteins that relieson their amphipatic character or other properties.Although it was not studied in great detail, it wasnoted during the analysis with Multicoil that severalof the large LEA-like proteins identified in this studycontained segments in their variable regions thatshowed high probabilities (up to 80%) of formingdimeric or trimeric coiled coils (results not shown).The properties of these hypothetical coiled coils andtheir relationship with the oligomerization-formingpotential of the C-terminal end is unknown butstresses the importance of continuing the character-ization of this new class of protein and group 3 LEA

proteins in general. As an alternative, these amphi-patic proteins may function by stabilizing and/orprotecting partially denaturated proteins or mem-branes through their hydrophobic interactions. Sucha function has been proposed for the amphipatica-helices present in group 2 LEA proteins (Close,1996).

For the moment, several indications suggest thatthe small chloroplastic LEA-like proteins (LEA3-L1and -L2) evolved from larger proteins in groupLEA3-L3 through a deletion of their variable region,and that this may have occurred independently inseveral plants after the divergence of monocots anddicots. This statement is based on the observationthat Arabidopsis genomic sequences and EST data-bases have only representatives of the large proteins(T10644, BAB10116). Initially, we believed that theArabidopsis protein COR15a (Lin and Thomashow,1992) was a homolog of proteins from the LEA3-L1and -L2 groups even if it shared little homology. Thiswas based on common properties such as similarsize, amino acid composition, chloroplast localiza-tion, and amphipatic character. On the other hand,this study suggests that COR15a does not contain theconserved C-terminal region and shares little identitywith the large Arabidopsis proteins. However, wecannot rule out the possibility that COR15a is a func-tional homolog of both LEA3-L1 and L2 groups ofproteins since the properties responsible for the func-tion of COR15a, LEA-L1 and L2 proteins are stillunknown. The results obtained by Artus et al. (1996)have shown that COR15am improves the freezingtolerance of chloroplast frozen in situ and of proto-plast frozen in vitro by inhibiting the formation of thehexagonal II phase, which is a major cause of mem-brane damage.

Ongoing genomic and EST sequencing projects willhelp to determine the exact number and characteris-tics of this new class of proteins in plants. Increasedsequence data may allow a correlation to emergebetween the appearance of the small proteins likeLEA3-L1 and -L2 and a phenotypic adaptation. It wasrather surprising that from the 243,000 rice (Oryzasativa), maize (Zea mays), and sorghum ESTs presentin GenBank, only two related ESTs were found. How-ever, previous Southern-blot experiments usingWcs19 as probe did not detect any signal in thecold-sensitive species corn and rice (result notshown). This suggests that cold-sensitive species maynot have evolved or else may have lost homologs ofLEA3-L1 and -L2 groups.

To investigate the function of LEA3-L2 group ofproteins, Wcs19 was constitutively expressed in Ara-bidopsis to determine whether it has a discernableeffect on tolerance to freezing and photoinhibition.The results presented in this study indicate that theobserved increase in freezing tolerance of cold-acclimated transgenic plants was statistically signifi-cant. However, it was not clear why the WCS19

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protein only improved the freezing tolerance of cold-acclimated plants. It is possible that WCS19 needsother chloroplast components or endogenous CORproteins that only accumulate in response to lowtemperature to accomplish its function, because itacts as part of a complex or in synergy with thesecomponents. WCS19 may interact with specific lipidsand/or proteins in the membrane and/or stroma.Therefore, if the(se) other component(s) accumulateslittle, if not at all, in 20°C grown plants, it couldexplain the absence of phenotypic effect on freezingtolerance as a result of the increased level of WCS19.

As a alternative, the WCS19 that is constitutivelyexpressed at 20°C could be non-functional. It mayonly become functional when activated in responseto a temperature shift (phosphorylation, pH-inducedconformational change, etc.). As mentioned previ-ously, one of the most important characteristics of theLEA3-L1, -L2, and -L3 groups of proteins is in theconserved C-terminal sequence. This region is pre-dicted to contain two different windows composed ofseveral heptad repeats that may be involved in atertiary interaction called a trimeric coiled coil. How-ever, it should be noted that a more precise designa-tion of the oligomerization ratio would have to awaitfurther work because it is known that Multicoil candetect other types of tertiary structures such as a foura-helical bundle (33% total probability) and a tet-rameric and pentameric coiled coils (54%; Wolf et al.,1997). This oligomerization potential raises the pos-sibility that homo-and hetero-oligomerization couldcreate a vast array of polypeptide complexes withdifferent or overlapping properties. In addition, theprediction that residues in the conserved C-terminalsequence can occupy two different positions in aheptad repeat raises the additional possibility thatthe segment may have the ability to shift to a secondstate based on environmental cues. Such a shift couldinduce a conformational change in the rest of thepolypeptide and hence, change the functional prop-erties of the protein complex. Although no experi-mental data exists to support this suggestion, it isknown that a drop in pH is instrumental in driving aregion of influenza hemaglutinin to adopt a coiledcoil structure provoking a conformational change(Carr and Kim, 1993). Thus, changes in chloroplaststromal pH or other modifications that occur duringexposure to low temperature may alter the structureof WCS19 and help to increase the freezing toleranceof plants.

In rye and wheat, increased freezing tolerance(LT50) was correlated with resistance to photoinhibi-tion (Pocock et al., 2001) and with the ability of plantsto maintain QA in a more oxidized state (Oquist andHuner, 1992; Oquist et al., 1993). Because the wheatLEA3-L2 protein (WCS19) was associated with anincrease in freezing tolerance of the transgenic Ara-bidopsis leaves and was localized to the chloroplast,we also determined whether the presence of WCS19

was associated with an increased resistance to pho-toinhibition. The preliminary results show that al-though the C71 line exhibited statistically (P , 0.05)increased resistance to photoinhibition, the transfor-mants shifted to low temperature exhibited generallyminimal changes in susceptibility to photoinhibitioncompared with wild type. Given that rye plants ex-hibited greater resistance to photoinhibition andexhibited much higher levels of WCS19 than the Ara-bidopsis transformants, it is possible that Arabidop-sis lines with higher levels of WCS19 expression maybe required to observe larger differences in resistanceto low-temperature photoinhibition. Regardless, thephotoinhibition results are consistent with the LT50data and confirm that there must be some additionallow temperature-induced factor required for WCS19to increase resistance to photoinhibition.

In summary, we have shown that the wheatLEA3-L2 (WCS19) is a stromal protein that belongs toa new class of organelle-targeted group 3 LEA pro-teins. The constitutive expression of the WCS19 pro-tein in Arabidopsis was shown to protect cold-acclimated leaves from freeze-induced damage.Despite the observed cryoprotective activity ofLEA3-L2 proteins, their exact roles are not clear. Fur-ther studies are required to determine their mode ofaction, to determine whether they act as part of acomplex, and to identify the chloroplast compo-nent(s) protected by this family of proteins duringstress conditions.

MATERIALS AND METHODS

Plant Material and Growth Conditions

Seeds of winter rye (Secale cereale L. cv Musketeer) and wheat (Triticumaestivum L. cv Fredrick and Norstar) were germinated in coarse vermiculiteand grown at temperatures of either 20/16°C or 5/5°C (day/night) with a16-h photoperiod in controlled environment chambers (Conviron, Mani-toba, Canada) as described previously (NDong et al., 2001). Growth irradi-ance was adjusted to 50 or 250 mmol m22 s21 at 5°C (5/50 and 5/250,respectively) and 50, 250, or 800 mmol m22 s21 at 20°C (20/50, 20/250, or20/800, respectively).

Growth conditions and LT50 (temperature at which 50% of the plants arekilled) determinations for the wheat and rye cultivars were as describedpreviously (Limin and Fowler, 1988). After 49 d of acclimation, the LT50

values were as follows: spring wheat cv Glenlea, 25.5°C; cv Manitou,26.2°C; and cv Chinese Spring, 29.4°C. Winter wheat cv Fredrick, 215.6°C;cv Besostoya, 216.4°C; cv Cheyenne, 219°C; cv Ulian, 219.5°C; and cvNorstar, 221.2°C. Rye cv Gazelle, 26.5°C; and cv Puma, 224.8°C.

Seeds from Arabidopsis wild-type plants (ecotype Columbia) and trans-genic Wcs19 lines (F3 populations) were germinated and grown in a mixcomposed of three parts Promix soil-less mix (Premier Brands, Riviere-du-Loup, Canada) and one part vermiculite. The plants were exposed to an 8-hphotoperiod with a growth irradiance of 100 mmol m22 s21. Plants that hadreached the fourth leaf stage were transferred to individual pots and grownuntil the appearance of 12 leaves at constant temperature of 20°C beforedetermining resistance to photoinhibition and freezing-induced cell dam-age. To examine the effects of low-temperature acclimation on both resis-tance parameters, plants that had been grown at 20°C were transferred to5°C for 7 d.

Plant Transformation

The Wcs19 cDNA was excised using SmaI and EcoRV that cut in thepolylinker region of pBluescript. The insert was ligated into the BamHI-SacI





restricted and Klenow-treated pBI121 vector between the CaMV 35S pro-moter and the nopaline synthase (NOS) terminator. The chimeric construct35SCaMV-Wcs19-NOS with the correct orientation was introduced intoAgrobacterium tumefaciens strain GV3101 (Koncz and Schell, 1986). The floraldip transformation protocol of Clough and Bent (1998) was used to trans-form Arabidopsis with A. tumefaciens carrying the construct.

Selection of putative transformants was performed as described byClough and Bent (1998) with slight modifications. To select for transfor-mants, sterilized seeds were suspended in 0.1% (w/v) sterile agarose andplated on 50 mg mL21 kanamycin and 500 mg mL21 cefotaxime. Threetransgenic lines (F0 population) that grew on kanamycin were transferred topots and moved into a growth chamber. A small leaf sample from thesetransgenic plants was tested for the presence of the WCS19 protein bywestern analysis. These three lines were advanced to the F3 population andwere found to be phenotypically uniform for kanamycin resistance andconstitutive production of WCS19 protein.

Assessment of Freezing Tolerance andResistance to Photoinhibition

The freezing tolerance of the transgenic Arabidopsis lines was estimatedusing the ion leakage technique as described previously (Gray et al., 1997;Pocock et al., 2001). Whole leaves were harvested from the plant, wrappedin moist cheesecloth, chilled rapidly to 22°C, and left for 1 h. Ice nucleationwas then induced with an ice chip. The leaf samples were then furthercooled at a rate of 2°C per hour to a minimum of 220°C and sampled at each2°C interval. The percent ion leakage was calculated as the ratio of theconductivity before and after boiling. The percent ion leakage was plottedversus temperature, and a sigmoidal function was fitted to the data usingthe Microcal Origin software package (Microcal Software Inc., Northamp-ton, MA). The temperature at which 50% of the total ion leakage occurredwas determined from the sigmoidal curve and used to estimate the LT50.

Photoinhibition of photosynthesis was induced by exposing detachedleaves of Arabidopsis to 1,600 mmol m22 s21 at 5°C. The photoinhibitorytreatment occurred under ambient O2 and CO2 conditions, and the leaveswere kept moist to minimize the effects of desiccation during the high-lightexposure. Estimates of PSII photochemistry (FV/FM) were used to monitorsusceptibility to photoinhibition. To measure FV/FM, the leaves were darkadapted for 10 min at room temperature and the F0 and FM values weredetermined using a Plant Stress Meter (PSM Chlorophyll Fluorometer,Biomonitor S.C.I. AB, Umeå, Sweden; Oquist and Wass, 1988).

Identification and Characterization of Rye and WheatGenes Sharing Identities with Wcs19

Recent analysis has revealed that nucleotides 1 to 285 of the originalWcs19 (Chauvin et al., 1993) are 98% identical to an unrelated EST and, thus,represent a fusion product during cDNA library construction. The GenBankfile has been corrected to reflect this new information.

Two strategies were used to identify genes related to Wcs19. As part ofour first strategy, three approaches were used to physically isolate homol-ogous genes. First, the complete Wcs19 cDNA (Chauvin et al., 1993) wasused to screen 100,000 plaques from the rye cDNA library prepared fromplants grown at 20°C and an irradiance of 800 mmol m22 s21 (20/800;NDong et al., 2001). Five plaques showing a strong hybridization signalwere selected and purified using standard molecular biology techniques(Sambrook et al., 1989). A clone of approximately 600 bases, Rep14 (for ryeexcitation pressure) was sequenced, and the deduced amino acid sequenceis presented in Figure 1. The gene was found to encode the rye ortholog ofWcs19.

Second, the rye 20/800 cDNA library was differentially screened withboth a 183-bp EcoRI-PstI fragment of Rep14 (encoding a putative chloroplasttransit peptide) and a 430-bp PstI-XhoI fragment. Eight plaques showing aspecific hybridization signal with the 183-bp fragment were selected, puri-fied, and analyzed by terminal sequencing. This revealed that five cloneswere identical and that only these clones showed a significant homologywith the chloroplast signal peptide. One of these, Rep13 was sequenced, andthe deduced protein sequence is presented in Figure 1. This Rep13 gene wasfound to encode the rye ortholog of Bcor14b, Wcor14a, and Wcor14b (Crosattiet al., 1999; Tsvetanov et al., 2000).

Finally, PCR was used to search for other wheat genes containing theputative chloroplast transit peptide. Poly(A1) RNA was isolated from 2-dcold-acclimated wheat cv Fredrick as described (Danyluk and Sarhan, 1990)and reverse transcribed with the first strand cDNA synthesis kit from RocheMolecular Biochemicals (Summerville, NJ) using the primer 59-GGCCAAG-CTTATCGATCC(T)17-39. PCR was performed using Taq DNA polymerase(Amersham Pharmacia Biotech, Uppsala) with the following primers: 59-GATGGCTTCTTCTTCCGTGCTGCTCG-39 and 59-GGCCAAGCTTATCGA-TCC-39. The PCR products were cloned into the pSTBlue-1 vector (Novagen,Madison, WI) using the Perfect Blunt Cloning kit (Novagen). Twenty-sevenclones with inserts were sequenced with the dye terminator sequencing kit(Beckman Coulter, Inc., Fullerton, CA) and run on a Beckman CEQ 2000sequencer. Sequencing revealed that 19 of them showed identities withWcor14a and Wcor14b, two were identical to Wcs19, and six were falsepositives and did not show any homology with the transit peptide. Clonesidentical to Wcor14a and similar to Wcor14b were sequenced. Their deducedprotein sequences are presented in Figure 1 under the names of WCOR14aand WCOR14c. This WCOR14c polypeptide differs from WCOR14b (Tsvet-anov et al., 2000) by six mismatches and does not contain the one-nucleotidedeletion that causes a frame shift mutation in the protein coding sequence.

In our second strategy, the nucleotide and the amino acid sequences ofisolated genes were subdivided into three segments (Fig. 1) and used tosearch the National Center for Biotechnology Information non-redundant,EST, and high-throughput genomic sequence databases with differentBLAST programs (Altschul et al., 1997). Sequences of genes or ESTs showinghigh scores were downloaded and analyzed in more detail. In the case ofESTs, several identical but overlapping ESTs were used to generate a morecomplete sequence for analysis.

Sequence analysis was done at sites such as the Canadian BioinformaticsResource (http://www.cbr.nrc.ca/), Biology Workbench (http://workbench.sdsc.edu/), and Expasy (http://www.expasy.ch/). Alignment tools usedwere ClustalW from BCM launcher analyses service (Baylor College ofMedicine search launcher; http://searchlauncher.bcm.tmc.edu) and Gap(http://www.cbr.nrc.ca/); programs for predicting subcellular localizationwere TargetP (Emanuelson et al., 2000), Predotar (http://www.inra.fr/internet/produits/predotar), ChloroP (Emanuelson et al., 1999), and Mito-prot (Claros and Vincens, 1996); and programs for analyzing secondary andtertiary structures were PELE (http://workbench.sdsc.edu/) and Multicoil(Wolf et al., 1997). Northern- and Southern-blot analyses were done asdescribed previously (Limin et al., 1997; NDong et al., 1997).

Production and Purification of WCS19 Antibodies

The Wcs19 cDNA was digested with MboI and subcloned into the BamHIsite of pTrcHisC (Invitrogen, Carlsbad, CA). The clone with the correctorientation was used to express Wcs19 as a N-terminal His-tagged fusionproduct in E. coli. The WCS19 protein was purified by affinity chromatog-raphy on a His-Bind resin (Novagen) and then separated on a 12% (w/v)SDS-polyacrylamide gel. The expressed protein was excised and electro-eluted for 3 h. Immune serum from rabbits injected with the WCS19 proteinwas found to cross-react with bacterial proteins. As a first step in purifyingthe specific WCS19 antibodies, the immune serum was first depleted ofthese cross-reacting antibodies. For this purpose, proteins from non-transformed bacteria eluting in the wash buffer (His-bind manual, Nova-gen) were concentrated using Centricon 10 (Amicon Inc., Beverly, MA),dialyzed against 120 mm HEPES (pH 7.5), and coupled at a concentration of2 mg mL21 with Affi-gel-10 beads in the presence of 80 mm CaCl2 (Bio-Radmanual, Hercules, CA). The immune serum was passed repeatedly on thiscolumn until monitoring showed no cross-reaction with bacterial proteins.The resulting immune serum was further purified on an affinity columncontaining Affi-gel-10 beads that were previously coupled to the WCS19protein at a concentration of 2 mg mL21 in the presence of 80 mm CaCl2.Specific anti-WCS19 antibodies were eluted with 0.1 m Gly (pH 2.5) andneutralized immediately with 1 m KPO4 (pH 8.0), concentrated using aCentricon 10 (Amicon Inc.), and frozen.

Protein Extraction and Immunoblot Analysis

Soluble proteins were extracted from frozen plant tissue as described(Houde et al., 1992b). To determine the boiling solubility, a fraction of thesupernatant was boiled for 10, 20, and 30 min and boiling soluble proteins

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were recovered by precipitation with 5 volumes of acetone and centrifuga-tion at 12,000g for 10 min. Proteins were separated on a 15% (w/v) SDS-polyacrylamide gel and transferred electrophoretically for 1 h at 100 V to a0.45-mm nitrocellulose membrane (Hybond-C; Amersham Pharmacia Bio-tech) without SDS in the transfer buffer. Immunoblotting was performed asdescribed previously (Danyluk et al., 1998) with the anti-WCS19 antibodydiluted at 1:10,000 and the secondary antibody diluted at 1:25,000.

Chloroplast Preparation and Fractionation

Chloroplasts were prepared using the method of Kunst et al. (1988) withslight modifications. Wheat leaves (10 g) were ground in 100 mL of extrac-tion buffer (0.45 m sorbitol, 20 mm Tricine KOH, pH 8.4, 2.5 mm EDTA, and5 mm MgCl2). The extract was filtered through two layers of Miracloth(Calbiochem, San Diego) and centrifuged at 270g for 90 s, and the pellet wasresuspended in buffer A (0.3 m sorbitol, 20 mm Tricine KOH, pH 7.6, 5 mmMgCl2, and 2.5 mm EDTA). The chloroplast suspension was then layered ona Percoll gradient previously prepared by centrifuging 50% (v/v) Percoll inbuffer A at 43,000g for 30 min in a SW41 Ti rotor. The gradients werecentrifuged at 13,000g for 6 min. Intact chloroplasts, which formed a bandnear the bottom of the gradient, were recovered, and an aliquot (wholechloroplasts) was mixed with 23 Laemmli buffer for analysis. The remain-ing intact chloroplasts were diluted with 1 volume of buffer A and pelletedat 3,000g for 90 s. The chloroplasts were then resuspended in buffer Awithout sorbitol and centrifuged at 3,000g for 5 min. The pellet (wholethylakoids) was resuspended in 13 Laemmli buffer for analysis. The super-natant constitutes the soluble fraction (stroma) and was precipitated with 5volumes of acetone, and the resulting pellet was suspended in 13 Laemmlibuffer for analysis.

Immunocytochemistry and Electron Microscopy

Discs 1.5 mm in diameter were cut from the fourth fully developed leafof plants grown at 20/250, 20/800, or 5/250, were fixed on ice using 0.5%(w/v) glutaraldehyde and 1.5% (w/v) paraformaldehyde in 0.2 m cacody-late buffer (pH 6.8) for 1 h, and were post-fixed in 2% (w/v) osmiumtetroxide. The samples were then rinsed with water and stained with 3%(w/v) uranyl acetate for 30 min. The leaf discs were dehydrated in a gradedethanol series and embedded in LR White resin.

Silver-gold sections were cut from the polymerized blocks using a SorvallMT2-B ultramicrotome equipped with a diamond knife and mounted onnickel grids (400 mesh). The specimens were then floated sample side downon droplets of the appropriate solutions for immunolabeling. The sectionswere etched with saturated sodium periodate for 8 min, washed with water,and treated with 0.1 n HCl for 10 min to further increase the availability ofantigenic sites (Craig and Goodchild, 1984). The sections were then blockedusing PTBN (0.02 m NaPO4 pH 7.4, 0.15 m NaCl, 0.1% [w/v] bovine serumalbumin, and 0.05% [w/v] Tween 20), and incubated overnight in anti-WCS19 antibodies at a dilution of 1:100 in PTBN. After washing in PTBN,the sections were incubated with 15-nm gold-labeled goat-anti-rabbit sec-ondary antibodies (Cedar Lane Laboratories) diluted 1:50 in PTBN for 30min. The specimens were then rinsed with water and post-stained with 3%(w/v) uranyl acetate before viewing. Photographic prints were made ofrepresentative chloroplasts from each treatment group. These were thenscanned into a digital format, and the number of gold particles per squaremicrometer of chloroplast was calculated using the northern Eclipse ImageAnalysis software package (v5.0, Emplix Image Inc., Mississagua, ON,Canada).

For the images presented, the 15-nm gold particles were enlarged usingsilver enhancement (Oliver, 1999). Enhancement was carried out for 5 minat room temperature in the dark. The samples were then rinsed thoroughlywith water, post-stained with 3% (w/v) uranyl acetate, washed with water,and then viewed.

To ensure the affinity and specificity of the WCS19 antibodies to the pureWCS19 protein, aliquots of the protein were placed on formvar-coatednickel grids (400 mesh), allowed to dry, blocked with PTBN, and thenincubated with the WCS19 antibody, the rabbit preimmune serum (both at1:100 dilutions), or distilled water for 10 min. The samples were thenwashed with PTBN, incubated with the gold-labeled goat-anti-rabbit sec-ondary antibody for 10 min, rinsed with water, and viewed.

To further ensure the specificity of our antibodies, sections of the 20/800grown plants were treated as described above, but the WCS19 antibody wassubstituted with either preimmune serum (1:100) or distilled water. Thus,ensuring that the rabbit had not been presensitized to chloroplast-localizedproteins, and that the secondary antibody was not binding directly to thespecimens.

Received January 4, 2002; returned for revision February 12, 2002; acceptedMarch 25, 2002.

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